Fluorescent proteins (FPs) have revolutionized biological research over the past two decades. Continued interest in engineering improved FPs has resulted in scores of FPs with unique properties tailor-fit to their experimental application. We are particularly interested in engineering next generation RFPs. In particular we are targeting bright, near-infrared (NIR) excitation and emission, which would allow for superior imaging in live animals, as biological tissue is maximally transparent to light in what is called the NIR window.
RFPs in their native form are not optimal molecular markers because of their obligate tetramerization, meaning that the cellular localization of any protein fused to a native RFP would be altered. Monomeric RFP variants have been engineered, most famously mCherry, by Roger Tsien and colleagues. There appears to be a limit to the brightness of these proteins however, and as RFPs are monomerized and their excitation and emission are evolved to far-red wavelengths, we and others have observed a commensurate drop in brightness.
We are interested in making brighter monomeric far-red proteins. With computational protein design tools we create libraries of RFP variants that we use to query mutational space in various structural regions of import. We guide the design of our libraries with biophysical constraints, evolutionary information, and experimental evidence.
Our lab has engineered many RFP derivatives over the years including: mRojo, mRouge, mPlumAYC, and the mGingers.
Sarah Gillespie - Graduate Student